Genes Similarly Altered by Treatment with GGF2 and EGF-domain only rhNRG in Post-MI Rats.

<p>Fold-changes in gene expression in treated compared to untreated animals are reported; negative value indicates downregulation in treated compared to controls.</p

Echocardiographic Measurements and Tissue Weight Ratios.

<p><i>FS%</i> fractional shortening, <i>LVIDd</i> left ventricular inner diameter at diastole, and <i>Heart/Body</i> heart weight to body weight ratio. Values are shown as predicted means with its 95% confidence interval. Groups are stratified by treatment and diet as follows: Normal Diet-Vehicle (n = 10), Normal Diet-Low Dose GGF2 (n = 9), Normal Diet-High Dose GGF2 (n = 9), Fatty Diet-Vehicle (n = 9), and Fatty Diet-High Dose GGF2 (n = 9). ^†FS % changes over time were analyzed using the generalized least squares linear regression method, and estimated means were fit using restricted maximum likelihood. Week 1 pre-treatment values were adjusted by adding a restricted cubic spline to the model. *Indicates significance with p<0.05 compared to vehicle animals. ^‡LVIDd changes over time were also analyzed using GLS regression fit by REML, and week 1 pre-treatment values were adjusted by restricted cubic spline. No significant difference was found between groups at each time point. ^§Heart/Body weight ratios were analyzed using ordinary least squares and one-way ANOVA. Heart/Body weight ratio comparison was found to be non-significant between groups.</p

Effect of GGF2 treatment on cardiac function early after MI.

<p>Serial measurements of fractional shortening (FS) were acquired from post-MI rats at baseline (1 week post-MI), 2 weeks after GGF2 treatment, and 4 weeks after GGF2 treatment. Echocardiographic assessment revealed that residual left ventricular (LV) FS% values were significantly higher (p = 0.0001) in GGF2 treated animals compared to those in the vehicle groups at the end of the study. The estimated mean FS% and range at 35 days post MI was 43.6 (41.0, 46.3) for the high dose treated group (n = 9), 42.0 (39.1, 45.0) for the low dose treated group (n = 9), and 36.6 (34.2, 39.1) for the vehicle group (n = 10. Individual rat FS % values trended downwards in the vehicle animals with time, whereas a progressive increase in FS % was observed in both the low dose and high dose GGF2 treatment groups. Moreover, the results indicate that high-fat feeding did not impair the effects of high dose GGF2 treatment on cardiac function.</p

Comparison of effects of GGF2 in rats early and late after MI.

<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055741#pone-0055741-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055741#pone-0055741-g002" target="_blank">2</a> are shown for direct comparison of effects of A) low dose and B) high dose GGF2 (mean +/− S.D.).</p

Examination of the effect of GGF2 on the myocardial proteome.

<p>Representative 2D gel image of total protein stain from the 5-gel coordinated DIGE experiment. A total of 300 µg of protein was loaded onto each DIGE gel. Altered proteins are indicated by numbers which correspond to line entries for identified proteins listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055741#pone-0055741-t003" target="_blank">Table 3</a>.</p

ErbB receptor protein expression in myocardium early and late after MI. A

<p>) Representative Western blot for ErbB2 and ErbB4 in early post-MI (5 weeks) vs late post-MI (12 weeks). <b>B</b>) Quantification of myocardial ErbB2 receptor expression after densitometric analyses. ErbB2 expression was normalized to α-tubulin expression for each of the groups. ErbB2 expression was increased early post MI compared to sham (p = 0.01) and late post MI (p = 0.03). <b>C</b>) Quantificationof myocardial ErbB4 receptor expression after densitometric analyses of blotted bands. Normalized ErbB4 expression to α-tubulin was similar in all groups.</p

Effects of GGF2 treatment on gene expression early after MI.

<p>A) Hierarchical clustering of RNA normalized fluorescent signal values was performed for 1,720 genes determined to be differentially expressed (p value <0.05, FC >1.5) in LV samples collected 4 weeks post-MI. Color indicates relative levels of expression replicates, with bright red indicating the highest fluorescent signal values and bright blue representing the lowest in the various samples shown. Each row represents one probe set, found as up- or down-regulated in GGF2-treated rats at 4 weeks post-MI. Columns represent the three replicates for each group, color-coded by sample type (Sham: pink; vehicle: yellow; GGF2: green). Two prominently differential clusters were generated that separated Sham-operated rats from those that received vehicle treatment. Two of the GGF2-treated samples were more similar to Sham-operated controls, indicating a normalization of MI-induced gene expression for these two biological replicates. This pattern was not detected for the third GGF2-treated sample, which clustered with vehicle-treated rats. B) GGF2 treatment in the two biological replicate ‘responders’ showed reversal of 853 genes altered by MI, compared to Sham-operated rats.</p

Altered Protein Expression in Post-MI Heart Muscle in GGF2-treated Rats Relative to Vehicle Controls.

+<p>Supported by protein, gene expression, and human repository data; ^‡supported by protein data only; conflicting results for protein and gene expression data ^§; nd  =  no measurable difference; <i>var</i> varying; Fold Differences (FD) in protein expression were considered as ‘altered’ only if of a magnitude of at least 1.3 (or 30% increase/decrease) was detected and p value was <0.05, based on one-way ANOVA. Ndufs1 and Bdh2 were one of several possible proteins that could represent the two respective spots. These two proteins were chosen based on their dual expression change based on proteomics and microarray analysis. However, it is possible that one of the other potential proteins, which included myosin 6 and myosin binding protein C, were instead representative of the two significant differential spots.</p

Effect of GGF2 treatment on cardiac function late after MI.

<p>A) Low dose GGF2: Rat FS % values were progressively reduced in both vehicle (n = 14) and low dose GGF-2 treated (n = 14) animals with time. The estimated mean FS% and range at 12 weeks post-MI was 32.9 (30.5, 35.3) for the low dose treated group and 31.7 (29.4, 33.9) for the vehicle group. Overall, the low dose treatment regimen did not restore late-post infarct LV structure and function because the average treatment effect was statistically insignificant (p = 0.51). B) High dose GGF2: FS % values decreased in vehicle (n = 4) animals and rose in high dose GGF2 treated (n = 8) rats with time. FS % was significantly increased in the high dose treated animals at 10,12,14, and 16 weeks post-MI (p = 0.013, p = 0.002, p = 0.002, and p = 0.017 respectively.) The estimated mean FS% and range at 16 weeks post-MI was 27.8 (24.1, 32.0) for the high dose treated group and 21.7 (18.1, 26.0) for the vehicle group. The average treatment effect of high dose GGF2 was found to be statistically significant (p = 0.005).</p

Effect of GGF2 treatment on myocardial PET glucose uptake.

<p>Short axis (transverse sections) images representative of sham, MI, and MI + GGF2 treated rats are displayed above. The sham rats showed prominent 18F-FDG uptake in all regions of the myocardium, whereas MI and MI + GGF2 treated rats show very little uptake in the infarct zone. Statistical analysis of cardiac standard uptake values was conducted by ANOVA, and results indicated that GGF2 treatment did not increase PET uptake in either remote or infarct regions (MI+LD, p = 0.79; MI+HD, p = 0.50).</p